PROJECT SUMMARY Cancer cells have evolved specific metabolic programs to support their growth and energetic demands. Decades ago, Otto Warburg described a mechanism by which cancer cells obtain intermediate metabolites for biomass from glucose in a process termed aerobic glycolysis. This ?Warburg effect? indicated that cancer cells preferentially convert glucose to lactate, even in the presence of oxygen. While inefficient in energy production, this process allows for the accumulation of metabolic intermediates used in lipid, protein, and nucleotide synthesis. Recent developments show that one cellular mechanism used to maintain ATP production during active proliferation is increasing the flux of glutamine into the TCA cycle (anaplerosis), a phenomenon particularly dominant in cancers driven by amplification of the oncogene Myc. How these tumors survive under conditions of limited glutamine remains unknown. This proposal describes an unbiased approach designed to discover novel molecular pathways that offer Myc-driven cancer cells a growth advantage, when challenged with glutamine depletion. Myc-amplified cells are highly dependent on glutamine in order to support their massive metabolic needs for growth and proliferation, as well as decrease oxidative stress. Glutamine depletion is highly toxic to these cells, suggesting that in order to survive, specific populations must develop unique metabolic adaptations. Furthermore, drugs intended to target glutamine metabolism for Myc-driven cancers are currently undergoing clinical trials. Anticipating that this targeting strategy proves successful, it is important to predict what novel metabolic adaptations these cells may acquire in response to prolonged treatment with glutamine metabolism inhibitors. This proposal aims to: First, analyze sequencing data from a glutamine-depleted screening strategy, and validate the top hits. Strikingly, in preliminary work we identified clones that not only survive glutamine withdrawal but also actively proliferate, suggesting that these cells are able to utilize alternative metabolic pathways to sustain growth. Second, validated hits will be metabolically characterized, using metabolic flux analyses and nutrient challenges to determine key metabolic pathways that have been reprogrammed to support active proliferation. Third, these pathways will be investigated in both ex vivo and in vivo models of Myc-driven cancer, to determine physiological relevance and feasibility of treatment development. This proposal's findings will enhance understanding of cellular metabolic reprogramming, and how it contributes to oncogenesis. Identification of these adaptations will not only provide mechanistic insights into novel metabolic pathways yet to be identified as key adaptive pathways in cancer cells, but could also lead to the development of the next generation of metabolism-targeted therapies for Myc-driven malignancies.